Opportunities in bile sampling for human ADME

Human ADME (Absorption, Distribution, Metabolism and Excretion) or ��mass balance�� studies are essential for understanding how investigational drugs behave in the body. These early phase studies typically involve a single dose of a radiolabelled compound, followed by collection of plasma, urine and faeces to determine excretion routes and metabolite profiles. The data confirm metabolite coverage in preclinical safety studies and identify any new metabolites in humans. The FDA, in its guidance document, recognises radiolabelled mass balance studies as ��single most direct method�� for gaining human ADME data¹.

However, traditional sampling leaves a blind spot: biliary excretion. Bile fluids are a major route for the elimination of drugs and metabolites in humans². It is often considered that bile sampling in humans is impractical, limiting insight into hepatic clearance and enterohepatic recirculation. Today, readily accessible and minimally invasive techniques are changing that.

Why bile sampling matters

Excreta sampling limited to urinary and faecal sampling provides overall excretion data, but this cannot distinguish between unabsorbed parent drug and drug material (parent drug and/or metabolites) secreted into bile after systemic exposure. Bile sampling offers direct evidence of hepatic elimination and recycling, which is critical for:

Drugs with high molecular weight or conjugated metabolites such as glucuronides and sulphates
Molecules subject to enterohepatic circulation, which can prolong exposure
Predicting drug�Cdrug interactions involving hepatic transporters

Without bile sampling, biliary contribution is inferred indirectly from faecal recovery, introducing uncertainty. In some cases, metabolites that appear negligible in faeces account for significant proportions in bile, highlighting the value of this matrix.

Additionally, small molecules are trending toward higher molecular weights³, meaning biliary sampling may become increasingly necessary in modern trials.

Challenges in human ADME studies

In nonclinical research, bile duct cannulation (BDC) in animals is a standard approach for collecting bile and understanding biliary clearance. This surgical method provides continuous bile flow for analysis but is not feasible in humans outside of surgical settings. The invasiveness of BDC and the associated medical and ethical considerations have historically prevented direct biliary assessment in early phase clinical trials, leaving sponsors to infer biliary contribution indirectly from faecal recovery.

Without direct bile or duodenal fluid sampling, investigators could not confirm whether faecal radioactivity represented unabsorbed drug or material secreted into bile. The result was a knowledge gap in mechanistic understanding of hepatic elimination pathways.

The solution lies in non?surgical, well?tolerated methods that integrate seamlessly into early phase studies.

Feasible methods for mass-balance bile sampling

Recent examples of alternative methodologies have proven successful, minimally invasive, and feasible: Entero-Tracker bile string and nasoduodenal tube sampling of duodenal fluids.

Bile string method

The bile string method (Entero?Tracker) is a gelatine capsule that houses a nylon string attached to a small steel weight. The volunteer swallows the capsule while the end of the string remains secured outside the mouth for retrieval. The capsule dissolves in the stomach and the string advances into the duodenum by peristalsis. On withdrawal the weight detaches at the gastric pylorus and is excreted naturally.

Typical use places the string two to three hours after dosing with radiolabel, keeps it in situ for four to four and a half hours, and triggers gallbladder contraction with a small food stimulus one hour before retrieval. Tolerability has been good across studies, including our own experiences, aligning with published clinical experience.

Advantages of the bile string method

The bile string poses minimal risk to the study participant, is unlikely to affect pharmacokinetics, and allows an entire cohort to be dosed on the same day, simplifying logistics and timelines. It can also be deployed more than once within a study if needed, for example with a baseline insertion and a post?dose insertion to contextualise change.

Nasoduodenal tube sampling

Nasoduodenal sampling is the more technical of the two methods. It uses a tube similar to standard enteral feeding devices, placed by a gastroenterologist with a pediatric gastroscope prior to dosing. Correct placement is confirmed by colour and pH of the first duodenal fluid aspirate.

The tube can remain in place for up to 11 hours post dose. Liquid meals are timed, e.g., at four and/or eight hours after dosing depending on the compound, to coincide with relevant expected excretion of parent drug and/or metabolites. This method has been integrated safely into mass balance designs and can generate time?resolved profiles of drug?related material in duodenal fluid.

Advantages of the nasoduodenal tube

Comparisons of subjects with and without a nasoduodenal tube have shown no meaningful impact on pharmacokinetics, and mass balance recovery remained within expected ranges.

The approach provides direct evidence of excretion of drug material into bile even when faecal recovery appears low. In one study a metabolite that contributed less than 1% of faecal radioactivity accounted for 10% of total radioactivity in duodenal samples⁴, highlighting the value of this matrix for mechanistic insight.

Study design and other considerations

Regulatory guidance for human radiolabelled mass balance requires at least six study completers. To de?risk delivery, 91�� recommends enrolling additional healthy volunteers.

For the bile string method, it is recommended to enrol seven or eight participants to maximise the chance of meeting the completer target. The procedure carries minimal risk and is unlikely to affect pharmacokinetics.

For nasoduodenal sampling, three design options are common, depending on sponsor preference, control comparison requirements, and operational logistics:

Enrol seven or eight participants and equip all with a nasoduodenal tube
Enrol six without a tube and four with a tube
Enrol four without a tube and four with a tube

The mixed?cohort designs enable comparison and, if needed, correction for any pharmacokinetic or metabolic shifts attributable to the tube while safeguarding the primary objectives of plasma pharmacokinetics, metabolite profiling, excretion routes and mass balance.

Operationally, nasoduodenal placement requires more time and scheduling considerations compared to bile string methods as the placement process is longer. Because a specialist gastroenterologist is required and scheduling is limited by their availability, sites may need to schedule across two subcohorts if four or more participants are involved. However, the resultant sample volume from the nasoduodenal tube is greater than with bile string, which enables detection of trace metabolites with greater confidence and accuracy.

Experienced partners for the new wave of ADME studies

91�� has assisted sponsors of all sizes with maximising the impact of their radiolabelled drug strategies for more than 25 years, including more than 200 studies generating key metabolic and drug-drug interaction data. To date, 91�� has successfully integrated bile sampling into ten human ADME studies��five using bile string and five using nasoduodenal tube. Studies employing these methods typically used conventional 14C doses between 3.0 and 3.7 MBq, with one study at 0.7 MBq due to dosimetry constraints.

Both methods were well tolerated, with no drop?outs due to procedures. Findings confirm that bile sampling adds critical clarity on biliary excretion and metabolite profiles without disrupting core study objectives.

Connect with us today to learn about our integrated approach to delivering fast, reliable ADME trials that help you meet regulatory requirements while optimising time and resources.

References:

¹U.S. Food and Drug Administration. Clinical pharmacology considerations for human radiolabeled mass balance studies: guidance for industry. Silver Spring, MD: U.S. Department of Health and Human Services; July 2024. https://www.fda.gov/media/158178/download

²Guiney WJ, Beaumont C, Thomas SR, Robertson DC, McHugh SM, Koch A, Richards D. Use of Entero Test, a simple approach for non invasive clinical evaluation of the biliary disposition of drugs. Br J Clin Pharmacol. 2011;72:133 142.

³Cuyckens F, Hvenegaard MG, Cassidy KC, Spracklin DK, James AD, Pedersen ML, Scarfe G, Wagner DS, Georgi K, Schulz SI, Schieferstein H, Bjornsdottir I, Romeo AA, Da?Violante G, Blech S, Moliner P, Young GC. Recommendations on the use of multiple labels in human mass balance studies. Drug Metab Dispos. 2024;52(3):153?158

⁴Remmerie B, Van den Boer M, Van Looy T, Wynant I, Rusch S, Huntjens D, De Meulder M, Stevens M. Integrating duodenal sampling in a human mass balance study to quantify the elimination pathways of JNJ?53718678, a respiratory syncytial virus fusion protein inhibitor. Adv Ther. 2020;37:578?591.

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